Anti col1a1

The effect of PF-05221304 to inhibit activation of hepatic stellate cells to a fibrogenic myofibroblast was assessed by measuring the effect of the compound on the production of αSMA and collagen from primary hepatic stellate cells stimulated with TGFβ1 to drive fibrogenesis. Briefly, human hepatic stellate cells (cat 5300; Sciencell Research Laboratories, Carlsbad, CA) were treated with 1 ng/mL TGFβ1 (cat 240B; R&D Systems, Minneapolis, MN) in the presence of varying concentrations of PF-05221304 or vehicle. Ascorbic acid was added at 100 μmol/L to the cell culture media to enhance the collagen production. Hepatic stellate cells unstimulated with TGFβ1 were used as a control. After a 72-hour incubation, the wells were methanol fixed, blocked, and incubated with anti-αSMA (clone 1A4; Sigma Aldrich) and anti-Col1A1 (cat C2456; Sigma), followed by matching secondary antibodies and Hoechst (Invitrogen, Carlsbad, CA) to produce an immunofluorescence signal. The cells were imaged on an Opera Phenix High Content Imaging platform (PerkinElmer). Images were analyzed with custom scripts in Columbus (PerkinElmer) for αSMA and collagen fluorescence intensity and nuclear counts. Effects on TGFβ1-stimulated proliferation was assessed by comparing the nuclear cell density of compound-treated cells to TGFβ1-stimulated vehicle-treated cells and the unstimulated control cells.

The following antibodies were used for immunostaining: anti-actin, α-smooth muscle (clone 1A4, A2547), anti-VCAN (polyclonal, HPA004726), anti-COL11A1 (polyclonal, HPA052246), anti-COL1A1 (polyclonal, HPA011795), anti-FN1 (polyclonal, F3648) all from Sigma-Aldrich, UK; anti-Ki67 (cloneMIB-1, M7240), from Dako, UK; anti-fibroblast activation protein, alpha (EPR 20021, ab207178), anti-COMP (ab11056), anti-CTSB (CA10, ab58802), anti-TGFβ all from Abcam; anti-PAX8 (NBP1-32440) from Novus; anti-EPCAM Alexa Fluor 488 conjugated (53-8326-41) from Thermo-Fisher; GLI-1 Antibody (C-1, sc-515751) from Santa Cruz Biotechnology.